Two powerful new technologies - siRNA-mediated gene silencing and high sensitivity mass spectrometry-now provide a powerful approach to the challenging problem of identifying novel insulin signaling proteins downstream of the PI 3-kinase pathway. We have successfully applied siRNA methodology to cultured 3T3-L1 adipocytes, demonstrating unequivocally the requirement of Akt2 protein kinase for insulin stimulation of GLUT4 translocation. The focus of this project is to discover and characterize protein substrates downstream of Akt that provide the missing links between insulin signaling and GLUT4 trafficking. We and others have recently discovered such novel candidate protein substrates using anti-Akt substrate phosphopeptide antibodies in combination with mass spectrometry. The aims of this proposed project include characterizing two of these substrates. The first is a novel Akt substrate we identified as a putative RIM binding protein (RIM-BP) that may regulate the GLUT4 exocytic process by interacting with a RIM or RIM-like protein in the plasma membrane. RIM1 controls exocytosis in neurons and RIM-BP1 appears to be a negative regulator of exocytosis. We show that RNAi-based depletion of adipocyte-expressed RIM-BP1 we identified by mass spectrometry enhances GLUT4 exocytosis in response to insulin. The second class of novel Akt substrates we shall characterize are two RabGAPs, one denoted AS 160 by the Lienhard laboratory, and an isoform (AS 160-2), that are hypothesized to regulate a Rab GTPase required for GLUT4-containing vesicle trafficking. We propose to identify the Rab protein target(s) of these novel RabGAPs through testing their GAP activities on a large set of Rab proteins assembled in vitro. Also, siRNA-mediated depletion of the AS160-1 and AS160-2 RabGAPs and of their Rab targets in 3T3-L1 adipocytes will be performed to test whether they function in GLUT4 regulation. Experiments to characterize the stages within the GLUT4 recycling pathway at which both RIM/RIM-BP proteins and RabGAP/Rabs operate will be performed. These studies will likely identify novel components of insulin signaling and GLUT4 regulation as well as reveal new mechanisms that mediate these processes.